SUMMARY Granulocyte-macrophage colony-stimulating factor (GM-CSF, CSF-2) is a pro-inflammatory cytokine that has emerged as the major pathogenic factor in autoimmune diseases, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). GM-CSF is required for the development and chronicity of EAE, and MS patients have increased numbers of GM-CSF-expressing B and T cells compared with healthy subjects. Both B and Th cells have been proposed as relevant sources of GM-CSF in EAE/MS, but it is currently unknown to what degree GM-CSF production by each of these cell types contributes to disease pathology. To address this question, we have developed GM-CSF conditional knockout mice, which have floxed GM-CSF gene (Csf2flox) that enable cell-specific ablation of GM-CSF. A cross between Csf2flox mice and those that express Cre recombinase in specific cell types will result in progeny lacking GM-CSF in those cells. We are crossing Csf2flox mice with Cre driver lines for Th1, Th17 and B lymphocytes in order to evaluate the role of GM- CSF derived from these cell types in EAE, thereby addressing a knowledge gap regarding the cellular sources of GM-CSF in autoimmune neuroinflammation. We propose the following specific aims: Specific Aim 1. To compare the relevance of Th1 and Th17 cells as the sources of GM-CSF in EAE. Although GM-CSF can be produced by most Th lineages, it is believed that its sources in EAE are Th1 and Th17 cells. We hypothesize that Th17 cells are the relevant cellular source of GM-CSF in EAE, while GM-CSF production by Th1 cells is dispensable. To test this, we have thus far generated mice lacking GM-CSF in Tbet- expressing cells (Th1 cells). We are currently finishing development of mice lacking GM-CSF in IL-17A- expressing cells (Th17 cells). These GM-CSF conditional knockout lines will be used to compare the relevance of Th1 and Th17 cells as the sources of GM-CSF in EAE. Specific Aim 2. To elucidate the role of GM-CSF from B cells in EAE. B cells play an important role in MS and in EAE, but their pathogenic mechanisms in these diseases are incompletely understood. Mouse and human B cells express GM-CSF, and MS patients have increased numbers of GM-CSF-producing B cells. It has been proposed that GM-CSF from B cells plays a pathogenic role in EAE/MS, but this hypothesis has not been tested, nor has GM-CSF production by B cells in EAE been characterized. Our preliminary data show that B cells, in the periphery and CNS, produce GM-CSF during EAE. We hypothesize that GM-CSF from B cells contributes significantly to EAE pathogenesis. To test this hypothesis, we currently generate mice that inducibly delete GM- CSF in B cells.